US7067622B2 - Method of the solid phase synthesis of pyrrole-imidazole polyamide - Google Patents

Method of the solid phase synthesis of pyrrole-imidazole polyamide Download PDF

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US7067622B2
US7067622B2 US10/481,275 US48127503A US7067622B2 US 7067622 B2 US7067622 B2 US 7067622B2 US 48127503 A US48127503 A US 48127503A US 7067622 B2 US7067622 B2 US 7067622B2
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pyrrole
fmoc
imidazole polyamide
carboxylic acid
imidazole
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US20040171799A1 (en
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Hiroshi Sugiyama
Hirokazu Iida
Isao Saito
Takashi Saito
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Japan Science and Technology Agency
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing three or more hetero rings

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  • the present invention relates to a novel method of synthesizing pyrrole-imidazole polyamide which is being noted as a useful compound for controlling a gene expression as a result of recent active researches which have clarified that the compound can be combined with a DNA in a sequence-specific manner.
  • the present inventors have focused and have been studying on distamycin, an antibiotic, which is known to selectively bond to a site of a DNA abundant with AT base pairs.
  • the inventors have synthesized systematic distamycin derivatives by combining a pyrrole amide which is a constituent unit of distamycin and an imidazole amide which is a derivative of the pyrrole amide and has been conducting biochemical researches on the thus-obtained compounds.
  • the inventors have clarified that each of the compounds is capable of selectively bonding with a specific site of DNA and the selectivity is defined by an order of pyrrole amide (Py) and imidazole amide (Im).
  • the inventors have found a systematic screening method for conducting a cytotoxicity test on the known cancer cells by using the synthesized pyrrole-imidazole polyamides.
  • This method is a simultaneous and easy screening method of the compounds targeting on DNA sequence specific to a certain cancer cell, which DNA sequence is being clarified in the human genome project.
  • the number of pyrrole-imidazole amides, which are the constituent units is 8, there are 256 combinations.
  • By screening those compounds simultaneously it is possible to systematically select cytotoxic compounds.
  • the system is innovative since it enables to derive a useful substance which uniquely suits a target gene from the DNA selective compounds (pyrrole-imidazole polyamides) of a countless combinations.
  • the present inventors have already filed a patent application of the invention (JP-A-2001-136974).
  • the method has a difficulty in synthesizing a polyamide having a carboxyl group at its end and is poor in efficiency in excising the end from the solid phase as a carboxylic acid residue, it is difficult to impart a new reactivity to the obtained long chain pyrrole-imidazole polyamide by direct modification.
  • the present invention has been accomplished in view of the above-described circumstances, and an object of the invention is to provide a method of producing a pyrrole-imidazole polyamide, whereby a longer pyrrole-imidazole polyamide can be conveniently synthesized and a protein (peptide) can be easily transferred; the method enables to efficiently produce a pyrrole-imidazole polyamide having a carboxylic acid residue at its end which can be excised from a solid phase carrier, making it possible to directly transfer various functional groups thereinto, and capable of exactly distinguishing DNA sequences.
  • the present invention provides a pyrrole-imidazole polyamide synthesis method characterized by performing automatic synthesis by the solid phase Fmoc method with the use of a peptide synthesizer.
  • the present invention provides a pyrrole-imidazole polyamide having a carboxyl group at its end.
  • the present invention provides a pyrrole-imidazole polyamide obtainable by transferring a DNA alkylation agent into the carboxyl group at the end of the above pyrrole-imidazole polyamide.
  • the present invention provides a sequence-specific DNA alkylation method characterized by using the above pyrrole-imidazole polyamide into which the DNA alkylation agent is transferred.
  • the present invention provides a FITC (fluorescein isothiocyanate)-pyrrole-imidazole polyamide conjugate having a carboxyl group at its end.
  • FITC fluorescein isothiocyanate
  • duocarmycin for example, it is possible to transfer duocarmycin, pyrrolo-benzodiazepin, bleomycin enediyne compounds, nitrogen mustard, and their derivatives, all having the DNA alkylation ability.
  • the method is the automatic synthesis using a commercially available protein (peptide) synthesizer, it is the synthesis method which enables a synthesis of a conjugate of a natural protein and a pyrrole-imidazole polyamide and a conjugate of a non-natural protein and a pyrrole-imidazole polyamide by automatic synthesis method.
  • reaction conditions of the Fmoc method are less strict than those of the t-BOC method, it is possible to transfer organic compounds, in addition to proteins, having a functional group which becomes unstable under the acidic condition and, therefore, the invention has a wider range of applicability.
  • the conventional liquid phase method for example, is not appropriate for synthesizing a different type, and the t-BOC method has a difficulty in synthesizing a long chain pyrrole-imidazole polyamide due to its strict reaction conditions as compared with those of the Fmoc method and is poor in applicability. Further, since the t-BOC method does not use any commercially available protein synthesizer, it also has a difficulty in transferring protein. Moreover, since the t-BOC method has a difficulty in excising the end from the solid phase as a carboxylic acid residue, it is difficult to impart a new reactivity to the obtained long chain pyrrole-imidazole polyamide by direct modification.
  • the synthesis of polypyrrole by the Fmoc method also has a problem of failing to exactly distinguishing DNA sequences, which is caused because an imidazole is not transferred into a DNA with the method; however, the method developed by the present inventors is an innovative method which solves all of the above problems.
  • FIG. 1 shows a result of analyzing a reactivity of a DNA alkylation agent to a DNA by polyacrylamide gel electrophoresis using a DNA fragment of 450 mer in a DNA alkylation experiment of Example 4(1).
  • FIG. 2 shows a result of analyzing a reactivity of a DNA alkylation agent to a DNA by polyacrylamide gel electrophoresis using a DNA fragment of 450 mer in a DNA alkylation experiment of Example 4(2).
  • FIG. 3 shows a result of analyzing a reactivity of a DNA alkylation agent to a DNA by polyacrylamide gel electrophoresis using a DNA fragment of 450 mer in a DNA alkylation experiment of Example 4(3).
  • pyrrole-imidazole polyamide having a carboxyl group at its end may be a pyrrole-imidazole polyamide having a ⁇ -alanine residue at its end, a pyrrole-imidazole polyamide having ⁇ -aminobutyric acid residue at its end, and the like.
  • the pyrrole-imidazole polyamide having a ⁇ -alanine residue at its end or the pyrrole-imidazole polyamide having a ⁇ -aminobutyric acid residue at its end may be synthesized by using an aminopyrrole carboxylic acid where the amino group is protected with Fmoc, an aminoimidazole carboxylic acid where the amino group is protected with Fmoc, and a solid phase carrier carrying ⁇ -alanine where the amino group is protected with Fmoc or ⁇ -aminobutyric acid where the amino group is protected with Fmoc and by an automatic synthesis by the solid phase Fmoc method using a peptide synthesizer.
  • aminopyrrole carboxylic acid may be 4-amino-2-pyrrole carboxylic acid, 4-amino-1-methyl-2-pyrrole carboxylic acid, 4-amino-1-ethyl-2-pyrrole carboxylic acid, 4-amino-1-propyl-2-pyrrole carboxylic acid, 4-amino-1-butyl-2-pyrrole carboxylic acid, and the like.
  • aminoimidazole carboxylic acid may be 4-amino-2-imidazole carboxylic acid, 4-amino-1-methyl-2-imidazole carboxylic acid, 4-amino-1-ethyl-2-imidazole carboxylic acid, 4-amino-1-propyl-2-imidazole carboxylic acid, 4-amino-1-butyl-2-imidazole carboxylic acid, and the like.
  • Reaction schemes of the syntheses of the aminopyrrole carboxylic acid where the amino group is protected with Fmoc and the aminoimidazole carboxylic acid where the amino group is protected with Fmoc in the case of using N-methylpyrrole or N-methylimidazole as a starting material by way of example are as follows.
  • a reaction scheme of the synthesis of ⁇ -aminobutyric acid where the amino group is protected with Fmoc is also shown below for reference.
  • the solid phase synthesis by the peptide synthesizer in the present invention is usually performed in a HATU[O-(7-azobenzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate]/DIEA (N,N-diisopropylethylamine) system.
  • Examples of the peptide synthesizer may be Pioneer (product of Applied Biosystems) which is a peptide synthesizer employing the Continuous Flow Method.
  • a reaction scheme of the solid phase synthesis of a pyrrole-imidazole polyamide of the present invention is as follows.
  • X natural and/or unnatural amino acid, and/or DNA and/or RNA examples of unnatural amino acid
  • an A-ring which is an active center of the DNA alkylation agent: DU-86 (2-methyl-3-methoxycarbonyl-A ring pyrrole-DUMA), is transferred into the carboxylic acid end excised from the solid phase carrier as follows.
  • a pyrrole-imidazole polyamide having a carboxylic acid end is dissolved into a solvent such as DMF, and then carbonyldiimidazole (CDI) is added to the mixture under a room temperature to be stirred overnight at the same temperature. After that, the solvent is distilled under a reduced pressure, and then the residue is washed with diethylether or the like to obtain an imidazole ester substance.
  • Du86 is dissolved into a solvent such as DMF, and then sodium hydride is added to the mixture under cooling (at ⁇ 15° C., for example), followed by stirring for 30 minutes.
  • a DMF solution containing the imidazole ester obtained above is dropped into the mixture, followed by stirring overnight at the same temperature.
  • a sodium phosphate buffer (pH6.86) or the like is added to the mixture to adjust pH, the solvent is distilled under a reduced pressure.
  • the obtained residue is purified by silica gel column chromatography and HPLC or the like to obtain a coupling substance (a pyrrole-imidazole polyamide into which the DNA alkylation agent is transferred).
  • a sequence-specific DNA alkylation becomes possible by the use of the thus-obtained pyrrole-imidazole polyamide into which the DNA alkylation agent is transferred.
  • FITC fluorescein isothiocyanate
  • Fmoc fluorescein isothiocyanate
  • the conjugate to be obtained is usable as a fluorescent labeling reagent which recognizes a specific DNA sequence and is capable of readily distinguishing DNA sequences relating to genetic diseases such as cancer.
  • Such conjugate can be used not only in a stage preceding a treatment with the pyrrole-imidazole polyamide, but also as a diagnostic drug.
  • the synthesis of the conjugate can be performed in the same manner as in the above-described solid phase synthesis of the pyrrole-imidazole polyamide, i.e., by the automatic synthesis by the solid phase Fmoc method with the use of the peptide synthesizer employing the Continuous Flow Method, such as Pioneer available from Applied Biosystems.
  • Monomer units 11, 12, and 14 to be used for the solid phase synthesis were synthesized by the following processes.
  • a commercially available protector was used as a Fmoc protector for ⁇ -alanine.
  • DMF dimethylformamide
  • DIEA diisopropylethylamine
  • TIS triisopropylsilane
  • TSA trifluoroacetic acid
  • CDI carbonyldiimidazole
  • DMAP 4-dimethylaminopyridine
  • a methylene chloride solution (200 ml) containing 1 (90.3 g, 1.10 mol) was added to a methylene chloride solution (600 ml) containing trichloroacetylchloride (200.0 g, 1.10 mol) by 3 hours' dripping. During the dripping, a nitrogen gas was sprayed so as to eliminate hydrogen chloride which is generated during the reaction. After stirring the mixture overnight, the solvent was distilled under a reduced pressure. The residue was subjected to silica gel column chromatography to obtain a trichloroacetyl substance 3 (189.7 g, 76%) from an elution position of ethyl acetate-hexane (1:10, v/v).
  • 10% palladium carbon (5 g) was added to a dichloromethane solution (300 ml) containing 8 (20.0 g, 108 mmol) to suspend the solution, followed by stirring the suspension for a day under a hydrogen atmosphere. After that, the suspension was filtered through a celite to remove palladium carbon, and then 10% hydrochloric acid was added to acidify the filtrate. Precipitates were collected by filtration to obtain 10 (19.8 g, 96%).
  • the solid phase synthesis is performed by using Pioneer (product of Applied Biosystems), which is a peptide synthesizer employing the Continuous Flow Method.
  • Used as a solid phase carrier was a commercially available Wang resin to which Fmoc- ⁇ -alanine is preloaded.
  • Four equivalent amounts of each of HATU, DIEA and the monomer units are used with respect to an active end of the solid phase carrier.
  • the solid phase carrier was swelled with DMF for 30 minutes, and then filled in a synthesis column of Pioneer. First, the Fmoc group of ⁇ -alanine preloaded to the carrier was deprotected by 5 minutes of treatment with the DMF solution containing 20% piperidine.
  • the carrier was washed with methanol for 50 seconds, and then the monomer unit, HATU, and DIEA to be transferred were passed through the column to be subjected to a cycling for 60 minutes, followed by washing again with methanol for 40 seconds.
  • the deprotection and the elongation were counted as one cycle, and the monomer units were condensed in accordance with the order of a sequence of a target polyamide.
  • the deprotection was performed in the same manner, and an acetylation was performed by using the DMF solution containing 5% acetic anhydride and 5% pyridine.
  • the solid phase carrier was taken out from the column to be subjected to a drying under reduced pressure and then moved to a 50 ml curved flask. 5 ml of the solution containing 95% TFA, 2.5% TIS, and 2.5% water was added to the solid phase carrier to be stirred for 30 minutes, whereby excising the polyamide from the carrier. Purification was performed by HPLC using a 0.1% TFA solution and acetonitrile.
  • Im represents a 1-methyl-4-aminoimidazole-2-carboxylic acid residue
  • Py represents a 1-methyl-4-aminopyrrole-2-carboxylic acid residue
  • Ac represents an acetyl group
  • ⁇ -butyl represents a ⁇ -aminobutyric acid residue
  • ⁇ -ala-COOH represents ⁇ -alanine in the following description.
  • the A ring (Du86) 15 which is the active center of DU-86 of the is the DNA alkylation agent is transferred into the carboxylic acid end excised from the solid phase carrier.
  • the operating procedure is as described below.
  • Carbonyldiimidazole (CDI, 24.3 mg, 0.15 mmol) was added to a DMF solution (1.5 mL) containing the pyrrole-imidazole polyamide (0.05 mmol) having the carboxylic acid end obtained in Example 2 under a room temperature, followed by stirring the mixture overnight.
  • the solvent was distilled under a reduced pressure, and the residue was washed twice with diethylether to obtain an imidazole ester substance (30 to 70%).
  • a DMF solution (2 mL) containing 15 (6.2 mg, 0.024 mmol) was cooled to ⁇ 15° C., followed by an addition of 60% sodium hydride (2.0 mg, 0.05 mmol), and then the mixture was stirred for 30 minutes at the same temperature. After dropping a DMF solution (1.5 mL) containing the imidazole ester obtained above (0.024 mmol), the mixture was stirred overnight at the same temperature. After adding sodium phosphate buffer (pH 6.86) to the mixture, the solvent was distilled under a reduced pressure. The obtained residue was subjected to silica gel column chromatography, followed by a purification by HPLC to obtain a coupling substance (20 to 50%).
  • Im′ represents an imidazolyl group
  • ⁇ -ala-CO represents a ⁇ -alanine residue
  • Ac, Im, Py, and ⁇ -butyl are the same as described above.
  • this compound alkylated the DNA with the hairpin structure in accordance with one rule. More specifically, a ⁇ -butyl hairpin curve portion recognized AT, a Py-Py pair recognized At or TA, and Du86 alkylated A or G.
  • the new molecular recognition pair ⁇ -alanine pair-Im pair selectively recognized a CG pair to reveal that it contributes significantly to the sequence-specific alkylation, which is the new discovery that have never been reported.
  • the synthesis was performed in the same manner as in the above-described solid phase synthesis of pyrrole-imidazole polyamide, i.e., by using Pioneer (product of Applied Biosystems) which is the peptide synthesizer employing the Continuous Flow Method.
  • Used as a solid phase carrier was a commercially available Wang resin to which Fmoc- ⁇ -alanine is preloaded.
  • Four equivalent amounts of each of HATU, DIEA, FITC and the monomer units are used with respect to an active end of the solid phase carrier.
  • the solid phase carrier was taken out from the column to be subjected to a drying under reduced pressure and then moved to a 50 ml curved flask.
  • a pyrrole-imidazole polyamide since it is possible to excise the end from a solid phase carrier as a carboxylic acid residue, various functional groups can be transferred into a pyrrole-imidazole polyamide. For example, it is possible to transfer duocarmycin, pyrrolo-benzodiazepin, bleomycine enediyne compounds, nitrogen mustard, and their derivatives, all having the DNA alkylation ability. In short, a compound which alkylates a DNA in a sequence-specific manner can readily be obtained by the method of the present invention. According to this technique, a progress in developments of drugs targeting DNAs specific to cancer cells, i.e., developments of anti-cancer drugs with less adverse side effects is greatly expected.
  • the present invention it is possible to synthesize a conjugate of a natural protein and a pyrrole-imidazole polyamide as well as a conjugate of a non-natural protein and a pyrrole-imidazole polyamide. Yet further, since the reaction conditions of the Fmoc method are less strict than those of the t-BOC method, it is possible to transfer organic compounds, in addition to the proteins, having a functional group which becomes unstable under a acidic condition and, therefore, the invention has a wider range of applicability. For example, it is possible to automatically synthesize the conjugates of a pyrrole-imidazole polyamide and DNA, RNA, or a derivative thereof.
  • conjugate by transferring FITC into a pyrrole-imidazole polyamide, and the conjugate to be obtained is usable as a fluorescent labeling reagent capable of recognizing a specific DNA sequence such as a DNA sequence relating to a genetic disease such as cancer.
  • conjugate can be used not only in a stage preceding a treatment with the pyrrole-imidazole polyamide, but also as a diagnostic drug.

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US20090171097A1 (en) * 2005-11-22 2009-07-02 Hiroshi Sugiyama Automated solid phase synthesis of pyrrole-imidazole polyamide
US20090306164A1 (en) * 2006-05-04 2009-12-10 Nanovir, Llc Methods for Treating Papilloma Virus Infection
US8674020B2 (en) 2009-04-28 2014-03-18 University Of Leicester Process for preparing polyamides
US10858646B2 (en) 2015-10-08 2020-12-08 Toppan Printing Co., Ltd. Method and kit for concentrating target double-stranded nucleic acid molecules using a pyrrole-imidazole-containing polyamide

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JP4682312B2 (ja) * 2003-09-04 2011-05-11 学校法人日本大学 ＴＧＦ−β遺伝子発現抑制剤
WO2005087762A1 (fr) * 2004-03-13 2005-09-22 Tmrc Co., Ltd. Nouveau dérivé indole servant à alkyler une sequence de bases spécifique d'adn et agent alkylant et médicament comprenant chacun celui-ci
JP2006022063A (ja) * 2004-07-09 2006-01-26 Univ Nihon Ｌｏｘ−１遺伝子発現抑制剤
JP2007176795A (ja) 2004-08-18 2007-07-12 Univ Nihon ＴＧＦ−β遺伝子発現抑制剤
US8993609B2 (en) 2006-05-04 2015-03-31 Nanovir, Llc Compounds for treating papilloma virus infection
JP5685081B2 (ja) * 2008-04-17 2015-03-18 浩喜 永瀬 マトリックスメタロプロテネース９遺伝子選択的発現抑制剤
EP2311494A4 (fr) * 2008-07-01 2012-09-05 Univ Nihon Inhibiteur de modification de l'histone spécifique d'un gène cible
RU2014113702A (ru) 2011-10-10 2015-11-20 НАНОВИР, ЭлЭлСи Гуанидинил-замещенные полиамиды, пригодные для лечения инфекций, вызванных папилломавирусом человека
US9982020B2 (en) 2011-10-10 2018-05-29 Nanovir Llc Antiviral compounds and methods for treating infections caused by double-stranded DNA viruses
US10350300B2 (en) 2013-10-11 2019-07-16 Chiba Prefecture Alkylating agent for alkylating target with driver oncogene mutation
EP3543233A4 (fr) 2016-09-21 2020-05-27 Chiba-Prefecture Nouvel agent d'alkylation
WO2023149487A1 (fr) * 2022-02-02 2023-08-10 国立大学法人千葉大学 Procédé de production de pyrrole-imidazole polyamide

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US20090171097A1 (en) * 2005-11-22 2009-07-02 Hiroshi Sugiyama Automated solid phase synthesis of pyrrole-imidazole polyamide
US20090306164A1 (en) * 2006-05-04 2009-12-10 Nanovir, Llc Methods for Treating Papilloma Virus Infection
US20100015084A1 (en) * 2006-05-04 2010-01-21 Nanovir, Llc Compounds for Treating Papilloma Virus Infection
US8119677B2 (en) 2006-05-04 2012-02-21 Nanovir, Llc Polyamides containing amino butyric acid-based building blocks
US9333232B2 (en) 2006-05-04 2016-05-10 Nanovir Llc Methods for treating papilloma virus infection
US8674020B2 (en) 2009-04-28 2014-03-18 University Of Leicester Process for preparing polyamides
US10858646B2 (en) 2015-10-08 2020-12-08 Toppan Printing Co., Ltd. Method and kit for concentrating target double-stranded nucleic acid molecules using a pyrrole-imidazole-containing polyamide
US11479764B2 (en) 2015-10-08 2022-10-25 Toppan Printing Co., Ltd. Method and kit for concentrating target double-stranded nucleic acid molecules using a pyrrole-imidazole-containing polyamide

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CA2450773C (fr) 2010-08-31
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